A cellulosic biomass is an effective starting material for a useful alcohol, such as ethanol, or an organic acid. In order to increase the amount of ethanol produced with the use of a cellulosic biomass, yeast strains capable of utilizing a xylose, which is a pentose, as a substrate have been developed. For example, Patent Literature 1 discloses a recombinant yeast strain resulting from incorporation of a xylose reductase gene and a xylitol dehydrogenase gene derived from Pichia stipitis and a xylulokinase gene derived from S. cerevisiae into its chromosome.
It is known that a large amount of acetic acid is contained in a hydrolysate of a cellulosic biomass and that acetic acid inhibits ethanol fermentation by a yeast strain. In the case of a yeast strain into which a xylose-assimilating gene has been introduced, in particular, acetic acid is known to inhibit ethanol fermentation carried out with the use of xylose as a saccharide source at a significant level (Non-Patent Literature 1 and 2).
A mash (moromi) resulting from fermentation of a cellulosic biomass saccharified with a cellulase is mainly composed of unfermented residue, poorly fermentable residue, enzymes, and fermenting microorganisms. Use of a mash-containing reaction solution for the subsequent fermentation process enables the reuse of fermenting microorganisms, reduction of the quantity of fermenting microorganisms to be introduced, and cost reduction. In such a case, however, acetic acid contained in the mash is simultaneously introduced, the concentration of acetic acid contained in a fermentation medium is increased as a consequence, and this may inhibit ethanol fermentation. In the case of a continuous fermentation technique in which the mash in a fermentation tank is transferred to a flash tank in which a reduced pressure level is maintained, ethanol is removed from the flash tank, and the mash is returned to the fermentation tank, although removal of acetic acid from the mash is difficult. Thus, inhibition of acetic acid-mediated fermentation would be critical.
In order to avoid inhibition of fermentation by acetic acid, there are reports concerning ethanol fermentation ability in the presence of acetic acid that has been improved by means of LPP1 or ENA1 gene overexpression (Non-Patent Literature 3) or FPS1 gene disruption (Non-Patent Literature 4) of Saccharomyces cerevisiae, which is a strain generally used for ethanol fermentation. However, such literature reports the results concerning ethanol fermentation conducted with the use of a glucose substrate, and the effects on ethanol fermentation conducted with the use of a xylose substrate, which is inhibited by acetic acid at a significant level, remain unknown. Even if the mutant yeast strains reported in such literature were used, the amount of acetic acid carry-over, which would be problematic at the time of the reuse of fermenting microorganisms or continuous fermentation, would not be reduced.
Alternatively, inhibition of fermentation by acetic acid may be avoided by metabolization of acetic acid in a medium simultaneously with ethanol fermentation. However, acetic acid metabolism is an aerobic reaction, which overlaps the metabolic pathway of ethanol. While acetic acid metabolism may be achieved by conducting fermentation under aerobic conditions, accordingly, ethanol as a target substance would also be metabolized.
As a means for metabolizing acetic acid under anaerobic conditions in which ethanol is not metabolized, assimilation of acetic acid achieved by introduction of the mhpF gene encoding acetaldehyde dehydrogenase (EC 1.2.1.10) into a Saccharomyces cerevisiae strain in which the GPD1 and GPD2 genes of the pathway of glycerine production had been destroyed has been reported (Non-Patent Literature 5 and Patent Literature 2). Acetaldehyde dehydrogenase catalyzes the reversible reaction described below.Acetaldehyde+NAD++coenzyme Aacetyl coenzyme A+NADH+H+
The pathway of glycerine production mediated by the GPD1 and GPD2 genes is a pathway that oxidizes excessive coenzyme NADH resulting from metabolism into NAD+, as shown in the following chemical reaction.0.5 glucose+NADH+H++ATP→glycerine+NAD++ADP+Pi
The reaction pathway is destructed by disrupting the GPD1 and GPD2 genes, excessive coenzyme NADH is supplied through introduction of mhpF, and the reaction proceeds as shown below.Acetyl coenzyme A+NADH+H+→acetaldehyde+NAD++coenzyme A
Acetyl coenzyme A is synthesized from acetic acid by acetyl-CoA synthetase, and acetaldehyde is converted into ethanol. Eventually, excessive coenzyme NADH is oxidized and acetic acid is metabolized, as shown in the following chemical reaction.Acetic acid+2NADH+2H++ATP→ethanol+NAD++AMP+Pi
As described above, it is necessary to destroy the glycerine pathway in order to impart acetic acid metabolizing ability to a yeast strain. However, the GPD1- and GPD2-disrupted strain is known to have significantly lowered fermentation ability, and utility at the industrial level is low. Neither Non-Patent Literature 5 nor Patent Literature 2 concerns the xylose-assimilating yeast strain, and, accordingly, whether or not the strain of interest would be effective at the time of xylose assimilation is unknown.
A strain resulting from introduction of the mhpF gene into a strain that was not subjected to GPD1 or GPD2 gene disruption has also been reported (Non-Patent Literature 6). While Non-Patent Literature 6 reports that the amount of acetic acid production is reduced upon introduction of the mhpF gene, it does not report that acetic acid in the medium would be reduced. In addition, Non-Patent Literature 6 does not relate to a xylose-assimilating yeast strain.
Also, there are reports concerning the introduction of the acetaldehyde dehydrogenase gene (derived from Entamoeba histolytica) into a xylose-assimilating yeast strain into which the xylose reductase (XYL1) gene (derived from Pichia stipitis), the xylitol dehydrogenase (XYL2) gene (derived from Pichia stipitis), and the xylulokinase (XKS1) gene (derived from Saccharomyces cerevisiae) had been introduced (Non-Patent Literature 7 and Patent Literature 3). According to such literatures, however, acetic acid assimilation did not take place at the time of xylose assimilation, and no difference was observed between a strain into which the acetaldehyde dehydrogenase gene had been introduced and a strain into which the acetaldehyde dehydrogenase gene had not been introduced in terms of acetic acid concentration in the medium.
According to conventional techniques, as described above, acetic acid would not be efficiently metabolized or degraded under conditions in which ethanol fermentation and xylose assimilation take place simultaneously.